Method for introducing foreign matters into living cells

a technology for living cells and foreign matter, applied in the field of methods for introducing foreign matter into living cells, can solve the problems of difficult introduction of foreign matter into a specified one cell of a specific tissue, and the expected dedifferentiation or redifferentiation system of many useful plants

Inactive Publication Date: 2006-11-07
OSAKA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0033]The laser to be used in the present invention is not particularly limited. The laser is excellent in that it has extremely excellent light-condensing ability, while giving almost no thermal influence upon a portion other than a laser beam spot. As the laser, the YAG laser, the exima laser, the Ar ion laser, the nitrogen laser, the nitrogen-excited color laser, etc. may be recited. The exima laser is preferred from the standpoint of view that a heat damage of the processed cell is particularly small, the processing accuracy is high, and the processed depth can be easily controlled by the irradiating energy and the number of times of irradiations.
[0095]When a antibacterial substance such as phytoalexin, more specifically, pisatin, phaseolin, medicarpin, rishitin or rishitinol is introduced likewise, disease tolerance of a tissue likely to be infected with pathogenic bacteria can be improved or completely sterilized cells free from viruses and bacteria can be prepared. In addition, tolerance against stress such as UV or light in a light-receiving tissue of a leaf or the like and that against stress such as a heavy metal in a root portion can be improved by incorporating an active oxygen such as phytochelatin or glutathione.

Problems solved by technology

Thus, molecular breeding of many useful plants of which dedifferentiation or re-differentiation system has not been established are expected.
From this point of view, there are problems of difficulties in introducing foreign matters other than genes in the case of a gene-introducing method utilizing agrobacteria carrying Ti plasmides, a protoplast fusion method and a gene gun method among the conventional foreign matter-introducing methods.
Further, a microinjection method, a liposome method and an electropolation method have a possibility of introducing foreign matters other than genes into cells, but the liposome method and the electropolation method require protoplast conversion when applied to plant cells, so that these methods have a problem in that there is difficulty in introducing a foreign matter into a specified one cell of a specific tissue.
The microinjection method has a problem in that there is extremely difficult because skill is needed to handle a plant cell having a cell wall tougher than that of an animal cell.
On the other hand, a laser method can introduce a foreign matter into a given target cell, but the conventional laser method has a problem in that there is difficulty in stably introducing foreign matters having extremely small physical strength, such as artificial chromosomes, or macrostructural bodies such as organella into cells.
However, such a method for introducing the foreign matter into the living cell has not been known yet.

Method used

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  • Method for introducing foreign matters into living cells
  • Method for introducing foreign matters into living cells
  • Method for introducing foreign matters into living cells

Examples

Experimental program
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Effect test

example 2

[0116]Next, a foreign matter was tried to be introduced into a cell by using the microinjection method after laser irradiation.

[0117]A hole, 10×10˜30×30 μm, was bored in aepidermiccell of an onion with the exima laser at an energy density of 30˜90 mJ / cm2. A calcein solution was introduced into the cell by using the microinjection through the hole bored in the laser processing. It was observed with the fluorescent microscope that the calcein solution was introduced into the cell.

example 3

[0118]Thirty days after a seed of a tobacco SR-1 plant was planted in a MS medium, a cotyledons was used and laser processed with an exima laser. The energy density of the laser was 30 to 90 mJ / cm2, and a hole was bored in a size of 10×10˜30×30 μm. A cell wall-digesting enzyme was dropped to the hole bored by the laser processing. Twenty four hours later, a protoplast cell of a mesophyll in which a cell wall near the hole was digested was isolated and observed.

example 4

[0119]Introduction of Liposome into a Cell

[0120]FITC-DEXTRAN

[0121]FLUORESCEIN ISOTHIOCYANETE-DEXTRAN (common name: FITC-DEXTRAN) commercially available from Sigma, Co., Ltd. was used. There was no limitation on the molecular weight of the FITC-DEXTRAN used.

[0122]Preparation of Liposome

[0123]Each of L-α-Dioleoyl Phosphatidyl ethanolamine and N-[1-(2,3-Dioleoyloxy)propyl]-N, N, N-trimethyl-ammonium was dissolved in chloroform at a concentration of 5 mg / ml. The resulting solutions were mixed at 200 μl:200 μl , and the chloroform was completely evaporated from the mixed solution at a low temperature under reduced pressure. A thin film of lipid was formed simultaneously with the evaporation. To this thin film was added 50 μl of a sample solution containing FITC-Dextran, which was left still on the film for 5 minutes. Then, 450 μl of a buffer solution: 10 mM Tris-HCl, pH 5.8 was added thereto, and left still for 15 minutes. The thin film of the lipid, the sample solution and the buffer so...

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Abstract

A method for introducing a foreign matter into a cell, includes the steps of placing a small particle carrying a foreign matter at a part of a cell surface of a living cell, boring a hole in a cell wall and / or a cell membrane by irradiating and treating said part of the cell surface with a laser beam, and introducing the foreign matter into the living cell.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a method for introducing a foreign matter into a living cell. Particularly, the invention relates to such a foreign matter into a living cell with use of a laser.[0003]2. Related Art Statement[0004]As the methods for introducing foreign matters such as genetic substances into living cells, there are known a method for directly introducing a foreign matter into a living cell with use of a fine glass tube (microinjection method), a method for applying a cell-fusing technique (a liposome fusion method and a protoplast fusion method), an introducing method using an infecting process (Agrobacterium method), a membrane permeability-promoting method (electropolation method), etc. when broadly classified.[0005]According to the microinjection method, a fine micropipette is prepared with use of a glass tube, connecting it with a micromanipulator, and introducing a liquid of a foreign matter into a...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N15/09A01H5/00C12N15/63C12N15/88C12N5/10C12M3/00C12N13/00C12N15/82C12N15/87
CPCC12M35/02C12N13/00C12N15/8206C12N15/87
Inventor KOBAYASHI, AKIOFUKUI, KIICHIHARAJIMA, SATOSHIFUKUSAKI, EIICHIROKAJIYAMA, SHINICHIROOKUDA, SHINYASHOJI, TAKESHI
Owner OSAKA UNIV
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